Ceramides form the backbone of all sphingolipids, and they have emerged as important regulators/mediators of cellular stress responses. The long-term goal of this proposal is to understand specific pathways of ceramide metabolism involved in cell stress response. Recently, significant complexity has emerged in ceramide metabolism and biology. Indeed, ceramides have emerged as a family of closely related molecules, the products of distinct metabolic pathways, with distinct sub-cellular localizations, and possibly mediating distinct functions. This recent realization that ceramides may not be 'equal' has necessitated a focus on individual pathways of ceramide metabolism that regulate cell stress. Work in our laboratory funded by three cycles of this grant led to the discovery that ceramides function as inducers of cell stress responses, the elucidation of some downstream mechanisms of ceramide-induced cell stress responses and apoptosis, and the discovery of subcellular pools of ceramides, including mitochondrial pools that are involved in cell death. Thus, recent work in our laboratory has focused on a newly identified family of enzymes, namely ceramide synthases (CerS), which in vitro demonstrate selective preferences for the fatty acyl chains that they incorporate into ceramide. In addition, very recent and exciting work in our laboratory has led to the discovery that CerS activity is selectively regulated by the pro-apoptotic Bcl-2 family member Bak. As such, key important questions emerge as to the specific roles of CerS enzymes in generating different ceramides in cells, their roles in recycling and subcellular location of ceramide in response to cell stress inducers, and, specifically, the mechanism of their regulation by Bak. To address these important questions we propose the following hypothesis: distinct CerS regulate the synthesis, recycling, metabolic fate and sub-cellular pools of individual ceramides in response to cell stress, and that Bak is a key regulator of CerS 5 and/or 6. To tackle this hypothesis, we propose the following specific aims: 1) To elucidate the roles of specific CerS in the synthesis, recycling, and metabolic fate of ceramides in cell stress responses. 2) To determine the roles of specific CerS in sub-cellular transport and distribution of ceramides in response to cell stress. And 3) To elucidate the mechanisms of regulation of CerS by the pro-apoptotic Bcl-2 family member Bak physiologically and in cell stress responses. Addressing these questions will provide key understanding of the function of specific CerS, will begin to implicate individual CerSs in cell stress responses, and study the mechanisms involved. Altogether, the proposed studies begin to unravel the emerging complexity of ceramide metabolism and function while making a novel breakthrough in the identification of a biochemical/molecular function for Bak.